Diterpenoids from Roots of Salvia lachnocalyx; In-silico and In-vitro Toxicity against Human Cancer Cell Lines

Further investigations on phytochemical constituents of dichloromethane extract from roots of Salvia lachnocalyx (S. lachnocalyx) led to the isolation and identification of eight known diterpenoids from this plant for the first time. The chemical structures of the purified compounds were elucidated using spectroscopic analyses including EI-MS, 1H and 13C NMR and by comparison of the resulting spectra with those reported in the literature. Then, the cytotoxic activity of identified compounds was examined against two human cancer cell lines MCF-7 (human breast adenocarcinoma) and K562 (human chronic myelogenous leukemia). Molecular docking of promising cytotoxic compounds were performed by AutoDock Tools 1.5.4 program in the active site of Topoisomerase I. Eight known diterpenoids; 12-hydroxysapriparaquinone (1), 15-deoxyfuerstione (2), horminon (3), 7α-acetoxyroyleanone (4), 11β-hydroxymanoyl oxide (5), microstegiol (6), 1-keto-aethiopinone (7) and 14-deoxycoleon U (8) were isolated of dichloromethane extract from roots of salvia lachnocalyx. Compounds 2, 3, 6, and 8 showed cytotoxic activity against MCF-7 (human breast adenocarcinoma) and K562 (human chronic myelogenous leukemia) cell lines with IC50 values in the range of 2.63-11.83 µg/mL. The inhibition of” topoisomerase I” was suggested by molecular docking calculations as the mechanism of cytotoxicity of the tested compounds. According to cytotoxic assay and docking results, it is suggested that compounds 2, 3, 6, and 8 have good potential as anticancer agents.


Introduction
Salvia (sage) is an important genus among the medicinal plants of the family Lamiaceae with about 900 species spread throughout the world. A literature survey demonstrated that Salvia species have been reported for treatment of many of different diseases. Abitane, rearranged abitane and tanshinones are principal secondary metabolites of the roots of Salvia species and reported with diverse pharmacological activities (1,2).
On continuing of our research on separation and structure elucidation of the bioactive compounds from Iranian Salvia species, the DCM extract of shoots and roots of S. lachnocalyx have been selected for further investigation based on primary results of cytotoxic screening of different Lamiaceae and Solanaceae plants (3).
We have subjected the shoots extract of S. lachnocalyx to a cytotoxic bioassay-guided fractionation experiments and separated two cytotoxic compounds; (2Z,6Z,10Z,14E)geranylfarnesol and spathulenol with IC 50 values ranging from 9.6 to 20.2 µg/mL against three human cancer cell lines (3). Recently, we have separated five diterpnoids including ferruginol, taxodione, sahandinone, 4-dehydrosalvilimbinol and labda-7,14-dien-13-ol with labdane, abietane, and rearrangedabietane diterpenoid skeletons from a DCM extract of S. lachnocalyx. The isolated compounds showed significant cytotoxicity against three human cancer cell lines (IC 50 range: 0.41-17.23 µg/mL) (7). In the present study we report more cytotoxic compounds from the polar fractions obtained from silica gel column chromatography of the DCM roots extract of S. lachnocalyx.
Assessment of the mode of action of natural and synthetic compounds by using computational approach is being increasingly exploited, because of this method being faster and cheaper than lab experiments (8). Docking studies is applied in both the rational drug design and mechanistic assessment by evaluating mode of interaction, binding affinity and orientation of a ligand into active site of the macromolecular target. Therefore, identification of therapeutic target has a key role in the discovery of new drugs (9).
In this study, in addition to isolation of further compounds from the roots extract of S. lachnocalyx, their cytotoxicity and possible mechanism of action have been examined by docking experiments.
Cell lines and culture MCF-7 (human breast adenocarcinoma) and K562 (human chronic myelogenous leukemia) cell lines were provided from Iranian Biological Resource Center, Tehran, Iran. The cells in monolayer were seeded in sterile T25 flasks in RPMI 1640 medium supplemented with 10% v/v fetal bovine serum, penicillin (100 units/mL) and streptomycin (100 µg/mL) and the flasks were incubated at 37 °C in a 5% CO 2 incubator.

Cytotoxicity assay
The cytotoxic activity was examined using the MTT reduction assay (10). In this colorimetric assay, the yellow color of tetrazolium bromide (MTT) is converted to the purple color of formazan by the action of mitochondrial dehydrogenase enzymes in viable cells. The dried compounds with suitable purity (≥ 95%) were dissolved in DMSO to obtain stock solution and then diluted in growth medium at least 400 times. The cells were added to each well of 96-well plates at the density of 50,000 cells/mL in 100 µL of growth medium and the plates were incubated at 37 °C for 24 h. After incubation, 50 µL of the medium was replaced with 50 µL of test compounds diluted in fresh growth medium (3-4 different concentrations) and incubation continued for a further 72 h. Then, the medium of each well was removed and replaced by RPMI without phenol red containing MTT 0.5 mg/mL and incubated for an additional 4 h. DMSO was used to solubilize the formed formazan crystals. The absorbance of the wells was measured at 570 nm, with background correction at 655 nm using a microplate reader and percentages of antiproliferative activity was calculated compared to the untreated control wells. IC 50 was calculated from the sigmoidal growth inhibition curves using CurveExpert software, version 1.3 for Windows.

Molecular docking study
Docking studies were carried out by AutoDock Tools 1.5.4 program (ADT) molecular simulation software. The X-ray crystallographic structure of the human DNA topoisomerase I (topo I) was downloaded from RCSB protein data bank (PDB code: 1k4t) for docking study. Co-crystallized ligand and water molecules were removed from crystal structures. Then, all polar hydrogen atoms were added and Kollman charges were assigned to the proteins and saved in pdbqt format by ADT. The grid map was determined based on the coordinates of native ligand (topotecan) in X-ray crystal structure. The energy minimized compounds were docked to active site of topo I using the Lamarkian genetic algorithm with grid sizes 60 × 60 × 60 (grid spacing 0.375 Å), the maximum number of evaluations were set to 2.5 × 10 6 , the number of GA runs were 100, and the maximum number of generations were set as 27,000, and all of the other options were set as default. For interpretation of docking result, the conformation with lowest binding free energies from the largest population cluster was selected.

Validation of docking
Performance and validation of docking study was evaluated by self-docking of the native ligand into the 1k4t active site. The selfdocking of native ligand into active pockets of receptor showed a binding free energies of −11.64 kcal/mol with RMSD of 0.846 Å. According to acceptable RMSD of selfdocking (< 2 Å), the obtained result indicates that the docking protocol was valid for topo I docking system.

Ligands preparation
The 3D structures of purified compounds were taken from PubChem data bank in SDF format (http://pubchem.ncbi.nlm.nih.gov). Gaussian 09 program was applied to minimize the ligands for docking purpose. Geometries of compounds were optimized using density functional theory (DFT) at B3LYP level of theory with the 6-31G (d) as general basis set in gas phase and the outputs of Gaussian were saved in pdb format. The vibrational frequency analysis was performed at the same level to check that there are no imaginary frequencies in minimized structures. Then, the gasteiger charges were added by ADT and saved in pdbqt format for docking study.

Calculation of molecular physicochemical properties
To evaluate purified compounds as drug candidate, some molecular properties such as octanol-water partition coefficient (log P), number of H-bond donors (HBD), number of H-bond acceptors (HBA), and molar refractivity (MR) were calculated using a freely accessible web-server (http://www.scfbio-iitd. res.in/software/drugdesign/lipinski.jsp) and also number of rotatable bonds (RB), and topological polar surface area (tPSA) were obtained from Pub Chem data bank for each compounds (Table 1).
The structure of the compounds were elucidated using the spectroscopic data including; EI-MS, 1 H and 13 1.61 and 1.77) which were in good agreement with those reported in the literature for 12-hydroxysapriparaquinone and 1-keto-aethiopinone, respectively (11,12). On the other hand, the NMR spectroscopic data of compounds 2, 3, 4 and 8 were quite similar to those reported for 15-deoxyfuerstione, horminon, 7α-acetoxyroyleanone, and 14-deoxycoleon U with an abietane diterpene characteristic signals. The quaternary methyl H(C)-20 signal appeared at δ H 1.56, 1.20, 1.23 and 1.67 ppm, for compounds 2, 3, 4, and 8 respectively. The rest recorded signals, especially the methyls signals of H(C)- [16][17][18][19] were in good agreements with those compounds that were isolated from Salvia moorcraftian (S. moorcraftian), Salvia sahendica (S. sahendica), Salvia rhytidea (S. rhytidea) and Salvia broussonetii (S. broussonetii) respectively (13)(14)(15)(16). In the process of structure elucidation of compound 5, the characteristic peaks of H(C)-14, 15 olefinic signal at δ H 5.85, 5.12, 4.91 and two methyls of H(C)-16, 17 at δ H 1.42, 1.59 ppm, respectively, together with the EIMS data guided us to the labdanetype diterpenoid which has been isolated from Salvia candidissima (S. candidissima), previously (17). Based on MS, 1 H and 13 C-NMR data, compound 6 was identified as a rearranged abietane diterpenoid that unlike the seco-4,5-abiatane a new cycloheptane ring was formed with a C-4/ C-11 bond formation, the resulting compound showed two germinal methayl signals at δ H 0.78, 0.79 H(C)-18, 19 were in agreement with the NMR data reported for microstegiol isolated from Salvia microstegia (S. microstegia), but upfield shifted in comparison to the respective signals of the abiatane diterpenoids (18). Compounds 2, 3, 5, 6, and 8 were tested against human breast adenocarcinoma (MCF-7) and human chronic myelogenous leukemia (K562) cell lines, while the remaining compounds were not tested because of the lack of quantity (Table 2). Several natural diterpenoids, especially abietane and labdan types have been investigated for their cytotoxic potential and have shown different levels of cytotoxicity against various cell lines. For instance, the cytotoxic activity of compounds 2, 5, and 8 against two selected cell lines and compounds 3 and 6 against K562 are being evaluated for the first time in the current study. Compound 2, 3, 6, and 8 exhibited cytotoxic effect with IC 50 values in the range of 2.63-11.83 µg/mL, which seemed as considerable activities as compared to cisplatin, a standard chemotherapeutic agent with IC 50 values of 12.49 and 2.91 µg/mL against MCF-7 and K562 cells, respectively. Compounds 2, 3, 6, and 8 showed higher cytotoxic activities compared to 5. The higher activity of the abietane diterpenoids (about 10 times greater) can be related to the presence of α,β-unsaturated carbonyl moiety in their structures or the higher cytotoxicity of abietane compared to labdane diterpenoids (19).
One of the most important drug targets in cancer therapy is topo I, which plays a unique role in DNA replication and cell division. Inhibition of topo I function results in DNA damage that ultimately leads to cell death. Therefore, topo I is considered as a therapeutic target for cancer chemotherapy (20). In a previous report, 324 natural compounds were screened for their capacity in topo I inhibition and among them 7-ketoroyleanone with abietane diterpenoid skeleton exhibited topo I inhibitory activities (21). Fronza and coworkers evaluated the inhibitory effects of some abietane diterpenoids such as 7α-acetoxyroyleanone, horminone, royleanone, 7-ketoroyleanone and sugiol on human DNA topo I. They showed that the abietane diterpenoids acted as topo I inhibitors in comparison to camptothecin, a positive control (22). According to the above mentioned report, topo I inhibition can be considered as possible mechanism for cytotoxicity of the purified compounds with abietane diterpenoids skeleton. Therefore, the above phenomenon is checked by molecular docking calculations.   Values are presented as mean ± SEM. of 3-5 experiments. Cisplatin was tested as a reference cytotoxic agent. Table 2. Anti-proliferative effects of diterpenoids isolated from roots of S. lachnocalyx against MCF-7 and K-562 cell lines.
Values are presented as mean ± SEM. of 3-5 experiments. Cisplatin was tested as a reference cytotoxic agent.  In order to assess the interactions of antiproliferative compounds with topo I; compounds 2, 3, 5, 6, and 8 were docked into the active pocket of topo I. Docking results of the purified compounds are presented in Table 1 as free energy of binding (ΔG b ) and K i (inhibition constant). Among the tested compounds, the compound 3 exhibited the best docking score with free binding energy of -8.30 kcal/mol and formed a hydrogen bond with a distance of 2.3 Å apart from the Asn722 amino acid residue of the topo I active site (Figure 2A). On the other hand, compound 6 demonstrated free binding energy of -8.22 kcal/mol and formed a hydrogen bond with DT10 in distance of 1.7 Å besides a stacking interaction with TGP11 ( Figure 2B). Compound 8 with free binding energy of -8.03 kcal/mol showed an H-bond with Thr718 at a distance of 1.9 Å besides two stacking with DA113 and TGP11 ( Figure 2C). 11-OH and 12 C=O groups in compound 2 formed two H-bonds with TGP11 at distances of 2.2 and 1.9 Å, respectively and caused a free binding energy of -8.01 kcal/mol. Different papers have demonstrated various orientations in active site of topo1 in interaction with their inhibitors (23)(24)(25)(26). These differentiations could be described based on molecular properties of topo I inhibitors. However, in all of them the H-bonding and stacking interaction between topo1 inhibitors with the DNA bases and critical residues of active site such as DA113, TGP11, Asn722, Lys532, Asp533, Arg364, Asn352, Tyr723 have been reported as the key interactions. Based on the obtained data, it was found that compounds 2, 3, 6, and 8 showed a good fitting in the active site of topo I, having docking scores between -8.01 to -8.30 kcal/ mol compared to compound 5 with the score of -6.93 kcal/mol.

Conclusion
The above mentioned docking results were in agreement with the order of higher cytotoxic activity of the isolated compounds, 2, 3, 6, and 8 with an abietane or rearranged carbon skeletons, while compound 5 with a labdane type skeleton has less cytotoxic activity. Therefore, based on docking results, the inhibition of topo I can be considered as a mechanism for the cytotoxicity of the tested active compounds.